1. Field of the Invention
The invention is in the field of stem cell growth and the modulation and maintenance of stem cell pluripotency. Specifically, small molecule mitochondrial respiration inhibitors are provided for the growth and manipulation of pluripotent stem cells under ambient oxygen conditions.
2. Description of the Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions or methods used in the present invention may be described in greater detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance or the prior art effect of the patents or publications described.
While the mechanisms underlying the maintenance of self-renewal and pluripotency are complex and poorly understood, there is evidence that culture conditions affect directly the growth and differentiation of stem cells. Ezashi et al. (Ezashi et al., 2005) demonstrated that culturing of human embryonic stem cells (i.e. hESCs) under low oxygen (O2) tension (5%) reduced the appearance of spontaneous differentiation. This may be the normal physiological state, as early-stage mammalian embryos also develop under low O2 concentrations (1.5%-5.3%) until they implant in the uterine endometrium, when O2 levels increase with vascularization (Fischer and Bavister, 1993).
When cultured under low O2 tension, mammalian cells decrease ATP production via oxidative phosphorylation in the mitochondria and increase glycolytic functions in order to meet energy demands. Studies of mitochondrial number and morphology in hESCs have demonstrated that undifferentiated hESCs have relatively few mitochondria in the cytoplasm and these mitochondria have few cristae, an indication of immature morphology (Cho et al., 2006; Oh et al., 2005; St John et al., 2005; Varum et al., 2011). As hESCs differentiate, the number of mitochondria with mature morphology increases, concomitant with the ATP levels produced by oxidative phosphorylation (Cho et al., 2006; Varum et al., 2011). Further maintenance of the pluripotent phenotype requires glycolysis (Zhou et al., 2012).
Moreover, it has previously been demonstrated that treatment with Antimycin A, an antibiotic isolated from streptomyces sp. which inhibits complex III of the mitochondrial respiratory chain, decreases ATP production via oxidative phosphorylation and simultaneously increases reactive oxygen species (ROS) formation in human embryonic stem cells. It also results in an increase in the expression of pluripotency factor NANOG and reduces differentiation (Varum et al., 2009). However, Antimycin A ([(2R,3S,6S,7R,8R)-3-[(3-formamido-2-hydroxybenzoyl)amino]-8-hexyl-2,6-dimethyl-4,9-dioxo-1,5-dioxonan-7-yl] 3-methylbutanoate) is known to be highly toxic (See U.S. Pat. No. 7,241,804) and accordingly undesirable for use in cell culture compositions that would contact human subjects.
US 20110301180, published Dec. 8, 2011, entitled “Reducing Platelet Activation, Aggregation and Platelet-Stimulated Thrombosis or Blood Coagulation by Reducing Mitochondrial Respiration,” by Collman et al. discloses that inhibiting mitochondrial respiration in platelets reduces platelet activation or platelet aggregation. Some compounds used there for inhibiting platelet activation have been found to be useful in the methods disclosed here. However, the platelet activation involves different cellular pathways than those of pluripotent stem cell differentiation.
Additionally, pluripotent stem cells have been produced by expression of core transcription factors in somatic cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) in a process called reprogramming resulting in induced pluripotent stem cells (iPS cells). These cells share many hallmarks with other pluripotent stem cells including preferential use of glycolytic pathways (Panopoulos et al., 2012; Varum et al., 2011).
It has also been demonstrated that inhibition of oxidative phosphorylation increases the efficiency of reprogramming to iPS cells (Son et al., 2013). In this study, Son et al. demonstrated that several inhibitors of oxidative phosphorylation were able to increase reprogramming efficiencies 10-20 fold and that there was demonstrated variability between inhibitors and their ability to affect reprogramming.
Mandal et al., “Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells,” Stem Cells. 2011 March; 29(3):486-95 disclose that attenuating mitochondrial function during the first 7 days of differentiation results in normal repression of Oct4, Nanog, and Sox2. However, differentiation potential is compromised as revealed by abnormal transcription of multiple Hox genes. The authors attenuated mitochondrial function in (a) self-renewing and (b) differentiating mouse and human ESCs using a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP).
Son et al. “Interference with the mitochondrial bioenergetics fuels reprogramming to pluripotency via facilitation of the glycolytic transition,” Int J Biochem Cell Biol. 2013 November; 45(11):2512-8 discloses that disturbance of mitochondrial metabolism by canonical mitochondrial inhibitors enhances metabolic reprogramming toward a glycolytic state, enabling the highly efficient generation of induced pluripotent stem cells. The authors used the mitochondria inhibitors rotenone, TTFA, antimycin A, KCN, oligomycin A and FCCP. However, these inhibitors are toxic. Rotenone exposure has been linked to the development of Parkinson's disease in farm workers. Rotenone also has off target effects on the microtubule cytoskeleton in addition to blocking the transfer of electrons from complex 1 to ubiquinone. Antimycin A is used as a piscicide, and as little as 200 mg is a lethal dose of KCN in humans.
There remains an unmet need to be able to grow and manipulate pluripotent stem cells, particularly human pluripotent stem cells, conveniently under ambient oxygen conditions.